Light-Induced Formation of Thymine-Containing Mercury(II)-Mediated Base Pairs

By applying caged thymidine residues, DNA duplexes were created in which Hg^II-mediated base pair formation can be triggered by irradiation with light. When a bidentate ligand was used as the complementary nucleobase, an unprecedented stepwise formation of different metal-mediated base pairs was achieved.     

Stable Copper(I)‐Mediated Base Pairing in DNA

A GNA (glycol nucleic acid) functionalized nucleoside analogue containing the artificial nucleobase 1H‐imidazo[4,5‐f][1,10]phenanthroline (P) was used to form a copper(I)‐mediated base pair within a DNA duplex. The geometrical constraints imposed by the artificial nucleobase play a pivotal role in this unprecedented stabilization of copper(I) in aqueous medium via metal‐mediated base pairing. The formation of the copper(I)‐mediated base pair was investigated by temperature‐dependent UV spectroscopy and CD spectroscopy. The metal‐mediated base pair stabilizes the DNA oligonucleotide duplex by 23 °C. A redox chemistry approach confirmed that this base pair formation was due to the incorporation of copper(I) into the duplex. This first report of a copper(I)‐mediated base pair adds metal‐based diversity to the field and consequently opens up the range of possible applications of metal‐modified nucleic acids.     

A Dinuclear Mercury(II)‐Mediated Base Pair in DNA

The first dinuclear metal‐mediated base pair containing divalent metal ions has been prepared. A combination of the neutral bis(monodentate) purine derivative 1,N⁶‐ethenoadenine (ϵA), which preferentially binds two metal ions with a parallel alignment of the N−M bonds, and the canonical nucleobase thymine (T), which readily deprotonates in the presence of Hg^II and thereby partially compensates the charge accumulation due to the two closely spaced divalent metal ions, yields the dinuclear T‐Hg^II₂‐ϵA base pair. This metal‐mediated base pair stabilizes the DNA oligonucleotide duplex as shown by an increase of 8 °C in its melting temperature. Formation of the base pair was demonstrated by temperature‐dependent UV spectroscopy as well as by titration experiments monitored by UV and CD spectroscopy.     

Metal‐Modified Nucleic Acids: Metal‐Mediated Base Pairs,Triples, and Tetrads

The incorporation of metal ions into nucleic acids by means of metal‐mediated base pairs represents a promising and prominent strategy for the site‐specific decoration of these self‐assembling supramolecules with metal‐based functionality. Over the past 20 years, numerous nucleoside surrogates have been introduced in this respect, broadening the metal scope by providing perfectly tailored metal‐binding sites. More recently, artificial nucleosides derived from natural purine or pyrimidine bases have moved into the focus of Ag^I‐mediated base pairing, due to their expected compatibility with regular Watson–Crick base pairs. This minireview summarizes these advances in metal‐mediated base pairing but also includes further recent progress in the field. Moreover, it addresses other aspects of metal‐modified nucleic acids, highlighting an expansion of the concept to metal‐mediated base triples (in triple helices and three‐way junctions) and metal‐mediated base tetrads (in quadruplexes). For all types of metal‐modified nucleic acids, proposed or accomplished applications are briefly mentioned, too.     

Sequence‐Dependent Duplex Stabilization upon Formation of a Metal‐Mediated Base Pair

An artificial nucleoside surrogate with 1H‐imidazo[4,5‐f][1,10]phenanthroline ( P ) acting as an aglycone has been introduced into DNA oligonucleotide duplexes. This nucleoside surrogate can act as a bidentate ligand, and so is useful in the context of metal‐mediated base pairs. Several duplexes involving a hetero base pair with an imidazole nucleoside have been investigated. The stability of DNA duplexes incorporating the respective Ag^I‐mediated base pairs strongly depends on the sequence context. Quantum mechanical/molecular mechanical (QM/MM) calculations have been performed in order to gain insight into the factors determining this sequence dependence. The results indicated that, in addition to the stabilizing effect that results from the formation of coordinative bonds, destabilizing effects may occur when the artificial base pair does not fit optimally into the surrounding B‐DNA duplex.     

Direct Observation of the Double-Stranded DNA Formation through Metal Ion-Mediated Base Pairing in the Nanoscale Structure

This work demonstrates single-molecule imaging of metal-ion induced double-stranded DNA formation in DNA nanostructures. The formation of the metal ion-mediated base pairing in a DNA origami frame was examined using C-Ag-C and T-Hg-T metallo-base pairs. The target DNA strands containing consecutive C or T were incorporated into the DNA frame, and the binding was controlled by the addition of metal ions. Double-stranded DNA formation was monitored by observing the structural changes in the incorporated DNA strands using high-speed atomic force microscopy (AFM). Using the T-Hg-T base pair, the dynamic formation of unique dsDNA and its dissociation were observed. The formation of an unusual shape of dsDNA with consecutive T-Hg-T base pairs was visualized in the designed nanoscale structure.     

Copper‐Induced Topology Switching and Thrombin Inhibition with Telomeric DNA G‐Quadruplexes

The topological diversity of DNA G‐quadruplexes may play a crucial role in its biological function. Reversible control over a specific folding topology was achieved by the synthesis of a chiral, glycol‐based pyridine ligand and its fourfold incorporation into human telomeric DNA by solid‐phase synthesis. Square‐planar coordination to a Cu^II ion led to the formation of a highly stabilizing intramolecular metal–base tetrad, substituting one G‐tetrad in the parent unimolecular G‐quadruplex. For the Tetrahymena telomeric repeat, Cu^II‐triggered switching from a hybrid‐dominated conformer mixture to an antiparallel topology was observed. Cu^II‐dependent control over a protein–G‐quadruplex interaction was shown for the thrombin–tba pair (tba=thrombin‐binding aptamer).     

Mechanistic Insight into the Photoinduced Damage of an Unnatural Base Pair

Chemical- and photostability of unnatural base pairs (UBPs) are important to maintain the genetic code integrity, and critical for developing healthy semisynthetic organisms. As reported, dTPT3 was less stable upon irradiation, and thus might act as a pervasive photosensitizer to induce oxidative damage within DNA, causing harm to living semi-synthetic organisms when exposed to UVA radiation. However, there was no knowledge about molecular-level understanding of this damage process. In this paper, we not only identified four photoproducts of dTPT3, including desulfur-dTPT3 (dTPT3^H), TPT3 sulphinate (TPT3^SO2), TPT3 sulphonate (TPT3^SO3) and TPT3-thioTPT3 (TPT3^STPT3), but also established a Type II photosensitized oxidation mechanism. In addition, the antioxidant (sodium ascorbate) was able to effectively inhibit the photoproducts formation of dTPT3 and dTPT3 in DNA, suggesting that a reductive environment might protect DNA bearing dTPT3 against UVA oxidation and ameliorate its adverse biological effects. The comprehensive understanding of TPT3’ photochemical stability will give researchers helpful guidance to design more photostable UBPs and construct healthier semisynthetic organisms.     

A Highly Stabilizing Silver(I)‐Mediated Base Pair in Parallel‐Stranded DNA

The first parallel‐stranded DNA duplex with Hoogsteen base pairing that readily incorporates an Ag⁺ ion into an internal mispair to form a metal‐mediated base pair has been created. Towards this end, the highly stabilizing 6 FP ‐Ag⁺‐ 6 FP base pair comprising the artificial nucleobase 6‐furylpurine ( 6 FP ) was devised. A combination of temperature‐dependent UV spectroscopy, CD spectroscopy, and DFT calculations was used to confirm the formation of this base pair. The nucleobase 6 FP is capable of forming metal‐mediated base pairs both by the Watson–Crick edge (i.e. in regular antiparallel‐stranded DNA) and by the Hoogsteen edge (i.e. in parallel‐stranded DNA), depending on the oligonucleotide sequence and the experimental conditions. The 6 FP ‐Ag⁺‐ 6 FP base pair within parallel‐stranded DNA is the most strongly stabilizing Ag⁺‐mediated base pair reported to date for any type of nucleic acid, with an increase in melting temperature of almost 15 °C upon the binding of one Ag⁺ ion.     

Covalently Cross-Linked Watson–Crick Base Pair Models

Structural characteristics of Watson–Crick hydrogen-bonded base pairs are displayed by methylene-bridged base pairs of type A . The shown superposition of the X-ray structure obtained for the base pair A (Rib¹=Et; Rib²=Me) over that of a C–G base pair illustrates that A occupies an area similar to that occupied by a traditional Watson–Crick hydrogen-bonded base pair. Temperature-dependent ¹H NMR studies indicate that the energy barrier for rotation along its CH₂ bridge is about 10 kcal mol⁻¹, and that it exists predominantly in one conformer at −70°C.     

Molecular Switching Behavior in Isosteric DNA Base Pairs

The structures and proton‐coupled behavior of adenine–thymine (A‐T) and a modified base pair containing a thymine isostere, adenine–difluorotoluene (A‐F), are studied in different solvents by dispersion‐corrected density functional theory. The stability of the canonical Watson–Crick base pair and the mismatched pair in various solvents with low and high dielectric constants is analyzed. It is demonstrated that A‐F base pairing is favored in solvents with low dielectric constant. The stabilization and conformational changes induced by protonation are also analyzed for the natural as well as the mismatched base pair. DNA sequences capable of changing their sequence conformation on protonation are used in the construction of pH‐based molecular switches. An acidic medium has a profound influence in stabilizing the isostere base pair. Such a large gain in stability on protonation leads to an interesting pH‐controlled molecular switch, which can be incorporated in a natural DNA tract.     

Small Sequence-Sensitive Compounds for Specific Recognition of the G⋅C Base Pair in DNA Minor Groove

A series of small diamidines with thiophene and modified N-alkylbenzimidazole σ-hole module represent specific binding to single G⋅C base pair (bp) DNA sequence. The variation of N-alkyl or aromatic rings were sensitive to microstructures of the DNA minor groove. Thirteen new compounds were synthesized to test their binding affinity and selectivity. The dicyanobenzimidazoles needed to synthesize the target diamidines were made via condensation/cyclization reactions of different aldehydes with different 3-amino-4-(alkyl- or phenyl-amino) benzonitriles. The final diamidines were synthesized using lithium bis-trimethylsilylamide (LiN[Si(CH₃)₃]₂) or Pinner methods. The newly synthesized compounds showed strong binding and selectivity to AAAGTTT compared to similar sequences AAATTT and AAAGCTTT investigated by several biophysical methods including biosensor-SPR, fluorescence spectroscopy, DNA thermal melting, ESI-MS spectrometry, circular dichroism, and molecular dynamics. The binding affinity results determined by fluorescence spectroscopy are in accordance with those obtained by biosensor-SPR. These small size single G⋅C bp highly specific binders extend the compound database for future biological applications.     

Solution Structure,Mechanism of Replication,and Optimization of an Unnatural Base Pair

As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge‐on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivatives of the nucleotide analogue d MMO2 , which forms an unnatural base pair with the nucleotide analogue d 5SICS . Replacing the para‐methyl substituent of d MMO2 with an annulated furan ring (yielding d FMO ) has a dramatically negative effect on replication, while replacing it with a methoxy (d DMO ) or with a thiomethyl group (d TMO ) improves replication in both steady‐state assays and during PCR amplification. Thus, d TMO –d 5SICS , and especially d DMO –d 5SICS , represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure–activity relationships governing unnatural base pair replication, we determined the solution structure of duplex DNA containing the parental d MMO2 –d 5SICS pair, and also used this structure to generate models of the derivative base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.     

Structure Determination of an AgI‐Mediated Cytosine–Cytosine Base Pair within DNA Duplex in Solution with 1H/15N/109Ag NMR Spectroscopy

The structure of an Ag^I‐mediated cytosine–cytosine base pair, C–Ag^I–C, was determined with NMR spectroscopy in solution. The observation of 1‐bond ¹⁵N‐¹⁰⁹Ag J‐coupling (¹J(¹⁵N,¹⁰⁹Ag): 83 and 84 Hz) recorded within the C–Ag^I–C base pair evidenced the N3–Ag^I–N3 linkage in C–Ag^I–C. The triplet resonances of the N4 atoms in C–Ag^I–C demonstrated that each exocyclic N4 atom exists as an amino group (?NH₂), and any isomerization and/or N4–Ag^I bonding can be excluded. The 3D structure of Ag^I–DNA complex determined with NOEs was classified as a B‐form conformation with a notable propeller twist of C–Ag^I–C (?18.3±3.0°). The ¹⁰⁹Ag NMR chemical shift of C‐Ag^I‐C was recorded for cytidine/Ag^I complex (δ(¹⁰⁹Ag): 442 ppm) to completed full NMR characterization of the metal linkage. The structural interpretation of NMR data with quantum mechanical calculations corroborated the structure of the C–Ag^I–C base pair.     

A Family of “Click” Nucleosides for Metal‐Mediated Base Pairing: Unravelling the Principles of Highly Stabilizing Metal‐Mediated Base Pairs

A family of artificial nucleosides has been developed by applying the Cu^I‐catalyzed Huisgen 1,3‐dipolar cycloaddition. Starting from 2‐deoxy‐β‐D ‐glycosyl azide as a common precursor, three bidentate nucleosides have been synthesized. The 1,2,3‐triazole involved in all three nucleobases is complemented by 1,2,4‐triazole ( TriTri ), pyrazole ( TriPyr ), or pyridine ( TriPy ). Molecular structures of two metal complexes indicate that metal‐mediated base pairs of TriPyr may not be fully planar. An investigation of DNA oligonucleotide duplexes comprising the new “click” nucleosides showed that they can bind Ag^I to form metal‐mediated base pairs. In particular the mispair formed from TriPy and the previously established imidazole nucleoside is significantly stabilized in the presence of Ag^I. A comparison of different oligonucleotide sequences allowed the determination of general factors involved in the stabilization of nucleic acids duplexes with metal‐mediated base pairs.     

Intramolecular Folding of PolyT Oligonucleotides Induced by Cooperative Binding of Silver(I) Ions

Ag⁺-bridged T-Ag⁺-T was recently discovered in a Ag⁺-DNA nanowire crystal, but it was reported that Ag⁺ had little to no affinity to T nucleobases and T-rich oligonucleotides in solution. Therefore, the binding mode for the formation of this type of novel metallo base pair in solution is elusive. Herein, we demonstrate that Ag⁺ can interact with polyT oligonucleotides once the concentration of Ag⁺ in solution exceeds a threshold value. The threshold value is independent of the concentration of the polyT oligonucleotide but is inversely proportional to the length of the polyT oligonucleotide. The polyT oligonucleotides are intramolecularly folded due to their positively cooperative formation and the stack of T-Ag⁺-T base pairs, resulting in the 5′- and 3′-ends being in close proximity to each other. The intramolecular Ag⁺-folded polyT oligonucleotide has a higher thermal stability than the duplex and can be reversibly modulated by cysteine.     

Bifacial Base‐Pairing Behaviors of 5‐Hydroxyuracil DNA Bases through Hydrogen Bonding and Metal Coordination

A novel bifacial ligand‐bearing nucleobase, 5‐hydroxyuracil ( U^OH ), which forms both a hydrogen‐bonded base pair ( U^OH –A) and a metal‐mediated base pair ( U^OH –M– U^OH ) has been developed. The U^OH –M– U^OH base pairs were quantitatively formed in the presence of lanthanide ions such as Gd^III when U^OH – U^OH pairs were consecutively incorporated into DNA duplexes. This result established metal‐assisted duplex stabilization as well as DNA‐templated assembly of lanthanide ions. Notably, a duplex possessing U^OH –A base pairs was destabilized by addition of Gd^III ions. This observation suggests that the hybridization behaviors of the U^OH ‐containing DNA strands are altered by metal complexation. Thus, the U^OH nucleobase with a bifacial base‐pairing property holds great promise as a component for metal‐responsive DNA materials.     

Metal-ion-dependent folding of a uranyl-specific DNAzyme: insight into function from fluorescence resonance energy transfer studies

Fluorescence resonance energy transfer (FRET) has been used to study the global folding of an uranyl (UO₂²⁺)‐specific 39E DNAzyme in the presence of Mg²⁺, Zn²⁺, Pb²⁺, or UO₂²⁺. At pH 5.5 and physiological ionic strength (100 mM Na⁺), two of the three stems in this DNAzyme folded into a compact structure in the presence of Mg²⁺ or Zn²⁺. However, no folding occurred in the presence of Pb²⁺ or UO₂²⁺; this is analogous to the “lock‐and‐key” catalysis mode first observed in the Pb²⁺‐specific 8–17 DNAzyme. However, Mg²⁺ and Zn²⁺ exert different effects on the 8–17 and 39E DNAzymes. Whereas Mg²⁺ or Zn²⁺‐dependent folding promoted 8–17 DNAzyme activity, the 39E DNAzyme folding induced by Mg²⁺ or Zn²⁺ inhibited UO₂²⁺‐specific activity. Group IIA series of metal ions (Mg²⁺, Ca²⁺, Sr²⁺) also caused global folding of the 39E DNAzyme, for which the apparent binding affinity between these metal ions and the DNAzyme decreases as the ionic radius of the metal ions increases. Because the ionic radius of Sr²⁺ (1.12 Å) is comparable to that of Pb²⁺ (1.20 Å), but contrary to Pb²⁺, Sr²⁺ induces the DNAzyme to fold under identical conditions, ionic size alone cannot account for the unique folding behaviors induced by Pb²⁺ and UO₂²⁺. Under low ionic strength (30 mM Na⁺), all four metal ions (Mg²⁺, Zn²⁺, Pb²⁺, and UO₂²⁺), caused 39E DNAzyme folding, suggesting that metal ions can neutralize the negative charge of DNA‐backbone phosphates in addition to playing specific catalytic roles. Mg²⁺ at low (<2 mM ) concentration promoted UO₂²⁺‐specific activity, whereas Mg²⁺ at high (>2 mM ) concentration inhibited the UO₂²⁺‐specific activity. Therefore, the lock‐and‐key mode of DNAzymes depends on ionic strength, and the 39E DNAzyme is in the lock‐and‐key mode only at ionic strengths of 100 mM or greater.     

Enhancing Phosphate Diester Cleavage by a Zinc Complex through Controlling Nucleophile Coordination

Metal‐ion complexes are the most effective artificial catalysts capable of cleaving phosphate diesters under mild aqueous conditions. A central strategy for making these complexes highly reactive has been to use ligand‐based alcohols that are coordinated to the ion, providing an ionised nucleophile under neutral conditions but at the expense of deactivating it. We have created a highly reactive Zn complex that is 350‐fold more reactive than an alcohol analogue by preventing the nucleophile binding to the metal ion. This strategy successfully delivers the benefits of efficient nucleophile delivery without strongly deactivating the metal ion Lewis acidity nor the oxyanion nucleophilicity. Varying the leaving group reveals that the transition state of the reaction is much further advanced than the reaction with hydroxide.